Microbial Ecology Driscoll Lecture 1 2024 PDF

MICR 331 Microbial Ecology Driscoll Lecture 1 1 1 Winter 2024 Course Information – In-person lectures for most, if not all, classes – myCourses - content – Course outline – PowerPoint files (pdf, 2 slides per page) – Lecture notes (outline) – Assignment materials – Submission via ‘Assignments’ on me...

MICR 331 Microbial Ecology Driscoll Lecture 1 1 1 Winter 2024 Course Information – In-person lectures for most, if not all, classes – myCourses - content – Course outline – PowerPoint files (pdf, 2 slides per page) – Lecture notes (outline) – Assignment materials – Submission via ‘Assignments’ on menu bar – Remote lectures (with recordings posted) only if in-person lectures not happening i.e., snowstorms, technical issues – Camtasia PowerPoint lectures possibly – Zoom lecture sessions, when needed – Office hours by appointment (contact me by email) 2 – Teaching assistant: Anthony Gagliano (contact by email) 2 1 Assessment o 15% Midterm #1: February 14, 2024, CCB o 20% Midterm #2: March 18, 2024, CCB o 10% Assignments: Four 1-page reports o Details to be announced in class o See course outline for mark breakdown and dates o Revisions must be submitted for Assignments 1 and 2 o 55% Final written exam: 3 hours, CCB – Date and time to be announced Textbook – none required – Optional: Atlas & Bartha – May become available on reserve, may be purchased on-line – Two scanned chapters available on myCourses 3 3 4 4 2 For this course: – You need to have some background in: – Genetics, evolution/phylogeny – Biochemistry – Microbiology – You do not need courses in all, but you need some – Prerequisites (one of): LSCI 230, AEBI 212, or ENVR 202, or permission of instructor – BSE (U2, U3) students should have enough relevant background from their U1 courses This is a 300-level course, not recommended for students in U1 – take it in U2 or U3 instead 5 5 Goals of this course: – To understand that microbial ecology is a field of research, done via both culture-based and molecular studies that has the goal of developing a better understanding of what complex natural populations of microbes do in natural environments – To consider microbial diversity, non-culturable organisms, and molecular phylogenetics in the context of microbial ecology – To appreciate the roles and physiological properties of microbes in various environmental processes (i.e., biogeochemical cycles) through the study of specific examples, including some from primary literature 6 6 3 Focus of this course: – Bacteria and Archaea – less emphasis on eukaryotic microbes such as fungi – the foundation of this course is how microbes interact with the environment and drive the biogeochemical cycles (C, N, S) – since we will cover these cycles throughout the course you will need to also read background material on your own – examples of the techniques that are being used to study different aspects of microbial ecology, especially molecular techniques – some topics will be covered to greater depth than others 7 7 Assignment #1 reading: Woese, C. R. et al., 1990. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences of the USA 87: 4576-4579. - Available via myCourses (Assignment materials) Why assign Woese paper? – to better understand what bacteria and archaea are – to understand where they fit into the universal phylogenetic tree – actual assignment questions later, for now, read it ASAP Why are we talking about phylogenetics and evolution? – critical for understanding modern microbial ecology 8 8 4 What is ecology? What is microbial ecology? 9 9 Ecology? – The study of organisms and their interactions with the environment and other organisms. – The study of the interrelationships between organisms and their biotic and abiotic environments Microbial ecology? – The study of the interactions between: – microbes - microbes, – microbes - macroscopic organisms, and – microbes - the environment – Microbial ecology is a fairly new and rapidly-changing field Microbial ecology is the study of the interactions between microbes and their biotic and abiotic environments 10 10 5 MICR 331 Microbial Ecology Driscoll Lecture 2 1 1 Assignment #1 - due January 26, 2024 One page – double spaced Woese, C. R. et al., 1990. Questions to be answered as part of the assignment: – What was the main point of the paper? – Why was this an important development? – What do you think of the paper (importance, readability)? Format and proofreading/revision process: Suggest three paragraphs: one per question – no numbers or headings: essay style. Spell-check and proofread, then have someone else proofread it. In turn, review their assignment. Revise your text based upon suggestions from your proofreader. Include your proofreader's name (as well as your own) on your paper. Submit a hard copy in class and submit to myCourses also. Revision: – Revise after you receive grade & comments and submit to myCourses 2 2 1 Extra references for Assignment #1 – Woese CR. Microbiol Rev. 1987 Jun;51(2):221-71. – Pace NR. Nature. 2006. May 18;441(7091):289. – Roberts E, Sethi A, Montoya J, Woese CR, Luthey-Schulten Z. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13953-8. – Woese CR, Goldenfeld N. Microbiol Mol Biol Rev. 2009 Mar;73(1):14-21. – Pace NR. Microbiol Mol Biol Rev. 2009 Dec;73(4):565-76. – Pace NR. J Bacteriol. 2009 Apr;191(7):2008-10. – Pace NR, Sapp J, Goldenfeld N. Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1011-8. 3 3 Environmental components important for microbes: elements Fig 11.1, from Wackett LP, Dodge AG, Ellis LB. Microbial genomics and the periodic table. Appl Environ Microbiol. 2004 Feb;70(2):647-55. 4 4 2 Environmental components important for microbes (life): – water: in liquid form – nutritional categories: energy, electrons, carbon – light: energy source for phototrophs, etc. – nutrients: C, N, P, S compounds, cations & anions, metals, etc. – for growth, metabolic functions – for generation of energy by chemotrophs – carbon source (autotrophs, heterotrophs) – something to oxidize AND something to reduce – electron donors and acceptors – for both chemotrophs and phototrophs – membrane potential, biosynthesis – surfaces to colonize (biofilms) – temperature, pressure, pH, [salt] 5 – etc… 5 Environmental components - electron donors and acceptors: – to energize membranes (membrane potential) for life – something to oxidize: an electron donor – organic (organotrophy i.e., carbohydrates CH2O, CH4) or – inorganic (lithotrophy i.e., H2, H2O, H2S, NH4+, Fe2+) – electrons removed from donor eventually pass to an electron acceptor during chemotrophy and phototrophy, or… – …are the source of electrons for biosynthetic reactions – electron donor may be energy source for chemotrophs, if energy is conserved in reactions that remove the electrons – something to reduce: an electron acceptor – organic (i.e., organic acids) or inorganic (i.e., O2, NO3, SO4, CO2, Fe3+, etc.) – during aerobic respiration, anaerobic respiration, 6 fermentation, phototrophy, methanogenesis, etc. 6 3 Summary of what microbes need to survive and grow: – liquid water – source of energy – something to oxidize and something to reduce – electron donor – electron acceptor – for membrane potential etc. – source of carbon – source of N, P, S, etc. 7 7 Metabolic energy: – phototrophs (light) or chemotrophs (chemical reactions) – energy needed to maintain membrane potential – for ATP biosynthesis, transport functions, etc. – energy and electrons needed to fuel metabolic reactions (biosynthesis, growth, etc.) – synthesis of amino acids, proteins – enzymes, structural proteins, etc. – synthesis of DNA, RNA – reproduction with fidelity, gene expression – synthesis of cellular structures i.e., cell walls 8 8 4 Early days of microbial ecology: – very difficult to study microbes in their natural environments – study first required isolation/cultivation of microbes from the environment as a pure culture – but most species (95-99%) were/are not culturable (or not yet?) Currently: – microbial ecologists use both culture-based and molecular methods to study complex microbial communities, from individual species to whole communities in situ – while a great deal of emphasis has been placed on the development of molecular tools in recent history, it remains essential to also continue to try to culture the new microbes identified via molecular techniques because molecular characterization alone is not enough, i.e., even to define/name a 9 new bacterial species 9 General objectives for the study of (microbial) populations: 1. 2. 3. 4. Detect Identify Quantify Characterize 10 10 5 General objectives for the study of (microbial) populations: 1. Detect – Detect the cells (microscope? PCR? metagenomic sequencing?), i.e., determine that microbes are present in the sample 2. Identify 3. Quantify 4. Characterize 11 11 General objectives for the study of (microbial) populations: 1. Detect – Detect the cells (microscope? PCR? metagenomic sequencing?), i.e., determine that microbes are present in the sample 2. Identify – Identify which organisms are present (to the phylum, genus, or species level, as appropriate), with or without cultivation 3. Quantify 4. Characterize 12 12 6 General objectives for the study of (microbial) populations: 1. Detect – Detect the cells (microscope? PCR? metagenomic sequencing?), i.e., determine that microbes are present in the sample 2. Identify – Identify which organisms (to the phylum, genus, or species level, as appropriate) are present, with or without cultivation 3. Quantify – Quantify the relative numbers of the different groups of microbes: population sizes, fluctuations over time or differences between conditions, etc. Must be done via molecular methods 4. Characterize 13 13 General objectives for the study of (microbial) populations: 1. Detect – Detect the cells (microscope? PCR? metagenomic sequencing?), i.e., determine that microbes are present in the sample 2. Identify – Identify which organisms (to the phylum, genus, or species level, as appropriate) are present, with or without cultivation 3. Quantify – Quantify the relative numbers of the different groups of microbes: population sizes, fluctuations over time or differences between conditions, etc. Must be done via molecular methods 4. Characterize – Characterize the physiological and ecological functions of different members of the population. This is currently the 14 biggest challenge! 14 7 General objectives for the study of (microbial) populations Challenges: – differentiation between live vs. dead, active vs. inactive cells —molecular techniques based on detection or amplification of DNA do not differentiate between living and dead cells or even ‘naked’ environmental DNA —detection or amplification of RNA (RT-PCR, transcriptomics) can be effective for investigating gene expression in active cells —microscopic techniques, even those using molecular probes (i.e., FISH), have difficulty differentiating living/dead and active/inactive cells —culture-based techniques can enrich for species that were not active, dominant or were just transiently in an environment —stable isotope probing (SIP) and other techniques are being developed to address the living/dead, active/inactive problems 15 15 8 MICR 331 Microbial Ecology Driscoll Lecture 3 1 1 Bring stromatolite and banded iron rock to class 2 2 1 What are microbes? 3 3 What are microbes? – Microscopic: – plants & animals (including protozoa) – bacteria – fungi – archaea – viruses & prions? – Are visible cells (including some bacteria) still ‘microbes’? Next questions: When and how did microbes (life) evolve? Why are microbes so diverse, widespread and abundant? 4 4 2 Timeline: 4.5 Billion-Years-Ago to Present 4.5 BYA - Earth formed, no free O2 in atmosphere (reducing) 4.5-4 BYA - crust cooled, rocks formed, oceans formed ~4 BYA - first microbes? - transition from chemical/biochemical to cellular life? - theory that the oceans were completely converted to steam on a number of occasions via meteorites up to ? BYA - periodic extinction & selection for thermophiles? ~3.5 BYA - cyanobacteria-like microfossils (Fig Tree formation, stromatolites). Earliest date still controversial 2.0 BYA 1.0 BYA - Gunflint formation (bacteria, cyanobacteria) - Bitter Springs formation (eucaryotic algae, fungi) 0.5 BYA 0.4 BYA Recently - marine invertebrates - insects - from 0.2 BYA: dinosaurs, mammals, etc 5 5 Evolution – time scale Invertebrates Unicellular algae Oxygenic cyanobacteria Aerobic bacteria Phototrophic bacteria Anaerobic cells then anaerobic bacteria, archaea, eucarya Oxic atmosphere 3.0 2.0 Billion years before present Sexual reporduction 4.0 Autotrophy Photosynthesis Chemical evolution 4.5 Diversity of life Hetertrophy Genomic life Formation of earth Anoxic atmosphere 1.0 0 6 3 7 7 Living stromatolites 8 8 4 Edmontosaurus (71-65 million years ago) 9 9 Cyanobacterial Palaeolyngbya microfossil (850 million years old) 10 5 Living cyanobacterium Oscillatoria 11 11 Microfossils of sulphurmetabolizing cells in 3.4-billionyear-old rocks of Western Australia Nature Geoscience 4, 698–702 (2011) 12 12 6 Microbial biotubules in 3.5 billion year old rock (lava/glass) known as 13 archaean pillow lavas (Furnes et al., Science, 2004) 13 Microbial excavation of solid carbonates Garcia-Pichel, F et al 2010 14 14 7 Evolution – time scale Invertebrates Unicellular algae Oxygenic cyanobacteria Aerobic bacteria Phototrophic bacteria Anaerobic cells then anaerobic bacteria, archaea, eucarya Oxic atmosphere 3.0 2.0 Billion years before present Sexual reporduction 4.0 Autotrophy Photosynthesis Chemical evolution 4.5 Diversity of life Hetertrophy Genomic life Formation of earth Anoxic atmosphere 1.0 0 15 Earth's atmosphere At formation: – high [CO2], low [N2] & [O2], some CH4, H2S, and maybe NH3 – reducing conditions, high temperature – greenhouse effect, but offset by lower solar flux, allowing liquid water to exist (eventually) Presently: – 0.03% CO2, 79% N2, 21% O2 – oxidizing conditions, 13oC – the atmosphere changed as a result of microbial activity – microbial activity (i.e., formation of organic compounds, O2) also changed terrestrial and aquatic environments – i.e., banded iron formations 16 16 8 Banded iron formation 17 17 Microbes have been evolving and exploiting niches for much longer than other organisms Therefore: – microbial life exists in "extreme" environments – microbes are extremely diverse – many "kingdoms" of bacteria and archaea – microbial taxonomic groups are deeply divergent – evolutionary distances between them are quite large Conclusions: – microbes literally created the earth's present environment – all life evolved from microbes and currently depends upon them – need to study microbial ecology to understand ecology in general 18 18 9 Microbes have been evolving and exploiting niches for much longer than other organisms Therefore: – microbial life exists in "extreme" environments – microbes are extremely diverse – many "kingdoms" of bacteria and archaea – microbial taxonomic groups are deeply divergent – evolutionary distances between them are quite large Conclusions: – microbes literally created the earth's present environment – all life evolved from microbes and currently depends upon them – need to study microbial ecology to understand ecology in general 19 19 Microbial Growth Potential Number of cells Log number of cells Review growth curves: – Lag phase: adjustment to new media before logarithmic growth – Log phase: exponential growth – Stationary phase: growth limited by depletion of nutrients, accumulation of waste products, cell death Time Stationary phase Log phase Lag phase Time 20 20 10 Microbial Growth Potential E. coli has a generation time of approximately 30 minutes under optimal conditions ! 30 min ! ¾¾® ! 1 30 min !! ¾¾® !! 2 30 min !!!! ¾¾® !!!! 3 etc 21 21 Microbial Growth Potential E. coli has a generation time of approximately 30 minutes under optimal conditions If growth was unlimited: – after 5 hours (10 doublings) – 103 cells (1024) 22 22 11 Microbial Growth Potential E. coli has a generation time of approximately 30 minutes under optimal conditions If growth was unlimited: – after 5 hours (10 doublings) – 103 cells (1024) – after 50 hours (~ 2 days) – 1030 cells – it has been estimated that there are in the range of 1030 cells on earth in total 23 23 If microbial growth was unlimited: – If all of the 1030 cells on earth were microbes, they would form a 20 cm-deep layer of cells over the entire planet's surface! – But this is not the case – Thus, microbial growth must be severely limited How? Why? 24 12 Microbial Growth Potential Conclusions: – most microbes are usually in a nutritionally-limited state – like stationary phase – extreme competition for limited resources – lots of cells get killed – unfit species disappear – better-fit mutant derivatives of these species may survive – less-fit mutants (the majority of mutants) less likely to survive if conditions do not change to favour them – these are the conditions (natural selection) that foster microbial evolution! 25 25 Actual number of cells on earth: – there are probably more than 1030 cells on earth – 1029 (est.) Prochlorococcus cells in the oceans – possibly 1030 bacterial & archaeal cells in soil, rock, sediments – thus, no one knows how many there really are, but 1030 would be relatively close Prochlorococcus marinus 26 26 13 Microbial cell walls – I will review the Gram stain technique, Gram-positive vs. Gramnegative bacteria vs. the cell walls and membranes of Archaea in very basic terms – review eucaryotic cell walls (cellulose, chitin, protein) on your own if you wish 27 27 14 MICR 331 Microbial Ecology Driscoll Lecture 4 1 1 Bring Winogradsky columns to class Movies: - The PCR Song, the PCR movie and the Translation movie will play on the desktop via VLC player or QuickTime - The PCR Song movie will play on the desktop via Windows Media Player 2 2 1 Microbial cell walls – Gram Stain – cytological characteristics were used for taxonomic purposes in the past but are not really valid for this purpose any longer – still interesting physiologically – Gram-positive and Gram-negative are two major cytological types of bacteria (but there are more types than these two) – the Gram stain can be useful to characterize bacterial cell walls (although it is imperfect) but is not useful for Archaea or Eucarya – other than the below Gram-positive groups, all bacterial groups that have been tested have been found to be Gram-negative – some Gram-negative groups (Deinococcus-Thermus) with very thick cell walls will stain false-positive with a Gram stain 3 3 Microbial cell walls – Gram stain – Gram-positive cell walls are found in four current bacterial groups (not monophyletic): 1. Firmicutes (including Bacillus sp.) – some Firmicutes stain Gram negative or have no cell walls (Mollicutes i.e., Mycoplasma) 2. Actinobacteria (including the Streptomycetes) 3. Chloroflexi – Chloroflexi mostly give a negative result in a Gram stain 4. Saccharibacteria (TM7 – one isolate reportedly cultured) 4 4 2 Woese's universal phylogenetic tree 1990: – Group 5 = Firmicutes plus Actinobacteria, which initially clustered together as the single Gram-positive group at the time – these were the only Gram-positive organisms known at the time 5 5 2016 Hug LA, et al. A new view of the tree of life. Nat Microbiol. 2016 Apr 11;1:16048 Gram +ve 6 6 3 7 Diagrams of the cell wall structure of Gram-negative (left) and Gram-positive bacteria. Key: peptidoglycan layer (yellow); protein (purple); teichoic acid (green); phospholipid ( brown); lipopolysaccharide (orange). 7 Gram negative Gram positive 8 8 4 Gram stain procedure 9 9 Archaea a | Schematic representation of a cross-section of the cell envelope of Sulfolobus solfataricus showing the cytoplasmic membrane, with membrane-spanning tetraether lipids and an S-layer composed of two proteins — a surface-covering protein (red oval) and a membrane-anchoring protein (yellow oblong). b | Schematic representation of a cell envelope of an archaeon that stains positive with the Gram stain and that contains a pseudomurein layer in addition to the S-layer. The cytoplasmic membrane is composed of diether lipids. 10 5 There are many different types of S-layers (or slayers) in Archaea and they are very different from bacterial surfaces 11 11 Methods for Studying Microbial Ecology 1. 2. 3. 4. Pure culture Microcosms Culture independent Genomics based 12 12 6 Pure-culture studies: – focus on one or a few species, in synthetic media – growth curves, physiological, genetic, medical studies – of limited relevance to the study of the ecological roles of that microbe, especially as most microbes cannot be grown in pure culture? 13 13 Microcosm studies: – natural habitat samples in the lab – Winogradsky columns are a type of microcosm – not pure cultures, but maintained under controlled conditions – manipulate parameters (i.e., temp., nutrients, radioactive tracers) – enrichment for certain organisms i.e., to enrich for N2-fixing bacteria use media with no N source other than air – monitor responses with or without manipulations i.e., – nutrient consumption, pollutant biodegradation, waste products, N2 fixation, CH4 oxidation, CO2 evolution, etc – use molecular biology tools to monitor presence of genes or gene products in populations – genes (DNA), gene expression (RNA) – could attempt to isolate pure cultures from the microcosm 14 14 7 15 15 Culture-independent (in situ) studies: – direct study of complex communities in environmental samples but with no culturing or enrichment – vs. time, depth, etc – Investigate: – where the microbes are (Detect) – which species (or phyla, etc) are present (Identify) – population sizes and fluctuations (Quantify) – physiological and ecological functions (Characterize) 16 16 8 How do we know that the sequences detected using molecular techniques represent real microbes? – they can be specifically visualized via FISH – microbiologists can sometimes coax them to grow in culture using their natural environment as a growth medium – understand that some species cannot survive on their own (obligate symbionts, organisms that depend on consortia of different species, etc) and so will never be isolated as pure cultures – some organisms have been nearly isolated via enrichment culture from environmental samples with thousands of species to mixed cultures with few species – promising technology currently being developed is the iChip 17 17 Enrichment and culturing of previously uncultured soil bacteria iChip Ling et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015 Jan 7. doi: 10.1038/nature14098. a–c, The iChip (a) consists of a central plate (b) which houses growing microorganisms, semi-permeable membranes on each side of the plate, which separate the plate from the environment, and two supporting side panels (c). The central plate and side panels have multiple matching through-holes. When the central plate is dipped into suspension of cells in molten agar, the through-holes capture small volumes of this suspension, which solidify in the form of small agar plugs. Alternatively, molten agar can be dispensed into the chambers. The membranes are attached and the iChip is then placed in soil from which the sample originated. 18 9 MICR 331 Microbial Ecology Driscoll Lecture 5 1 1 Bring iron pyrite to class Bring Winogradsky column 2 2 1 Culture-independent methods: – measurement of gasses (CO2, H2S, N2O, CH4 etc) and other metabolites, often using radioactive isotope tracers – molecular biological methods (many) – microscopy (many methods) – FISH (fluorescent in situ hybridization) microscopy to visualize specific cells, CARD-FISH – stable isotope probing (SIP) studies i.e., DNA-SIP 3 3 FISH of beewolf symbionts (Streptomyces) 4 4 2 Examples of some molecular biological techniques used in culture-independent studies: – extraction of nucleic acids (DNA, RNA) – genes relevant to phylogenetic (16S rRNA genes) or physiological (functional genes that encode enzymes) questions are often targeted in ecological studies – amplification of DNA sequences (genes) via PCR – PCR, DNA sequencing, qRT-PCR – hybridization-based techniques (blots, arrays, FISH, etc), – separation of DNA fragments by agarose or polyacrylamide gel electrophoresis – denaturing gradient gel electrophoresis (DGGE) – cloning of amplified genes for further study 5 5 Genomics, transcriptomics and bioinformatics studies: – many applications of genomics information can be used in microbial ecology today – new genome sequences can be obtained and compared to the large databases of genome sequences – ‘big-data’ bioinformatic analysis – microarrays – metagenomics – environmental nucleic acid sequencing – DNA = metagenomic sequencing – RNA = metatranscriptomics 6 6 3 Metagenomics: – in metagenomics studies, DNA from a population containing many species can be analysed and used in experiments – can sequence everything or focus on a few genes from a population, i.e., 16S rRNA for community profiling – have been done for many environments including: – polluted soils and ground water, wastewater treatment plants – drinking water, sea water, soils – biofilms on corroded oil pipelines – the bovine rumen – coastal RNA virus communities, the viruses of archaea – FAQ: The Human Microbiome: – https://www.ncbi.nlm.nih.gov/books/NBK562894/ – Venter, J.C. et al. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304(5667):66-74. 7 7 Historical Figures Relevant to Microbial Ecology Roger Knowles: microbial ecology, N cycle (nitrification, denitrification), C cycle (ammonium oxidation, etc), one of the first microbial ecologists in Canada, offered one of the first microbial ecology courses in Canada (MICR 331) 8 8 4 Louis Pasteur: – made many discoveries, but, most relevant to our discussion, demonstrated in a series of publications (1860-1862) that the theory of spontaneous generation of microorganisms (germs) was false 9 9 Pasteur experiment 10 10 5 Robert Koch: – Koch’s Postulates (late 19th century) 1. 2. 3. 4. The microorganism must be found in abundance in all organisms with the disease, but not in healthy ones. A pure culture of the microorganism must be isolated from a diseased organism. The cultured microorganism must cause the same disease when infected to a healthy host. The microorganism must be re-isolated from the experimentally-inoculated diseased host and be shown to be identical to the original isolate. 11 11 Sergei Winogradsky: – from the late 19th century to the early 1900's did the pioneering studies in chemoautotrophy, enrichment culture technique, and N2-fixation 12 12 6 Winogradsky column MOVIE Purple bacteria Green bacteria 13 13 7 MICR 331 Microbial Ecology Driscoll Lecture 6 1 1 Bring iron pyrite to class 2 2 1 Martinus Beijerinck: – in the early 1900's laid the foundation for our present understanding of N and S cycling by microbes, and N2 fixation – called himself a "microbial ecologist" 3 3 Alexander Fleming: – in 1929 did the ground-work in the discovery of antibiotics (penicillin), a fascinating microbe-microbe interaction – his prediction that the use of antibiotics would give rise to antibiotic resistant strains, based upon observations of spontaneous resistance, is still being ignored by many today 4 4 2 1950s – 1970s – huge advances in the study and understanding of biology – structure of DNA – basic cell biology, genetics, biochemistry – medical microbiology, virology, immunology – microbial taxonomy was based upon cytological and physiological factors – molecular genetics grew as a fusion of biochemistry and genetics – era of molecular biology begins – computers – microbial ecology continued to develop and incorporate new techniques as they emerged – pioneering work on biogeochemical cycles 5 5 Modern Era Explosive synergy in late 20th century (to present): – there was a general explosion in the understanding of biology/biochemistry, microbiology, bacterial molecular genetics, and rapid development of molecular biology (cloning, DNA sequencing etc), computer technology/science/math/stats (software and hardware) and the internet (bioinformatics) Synergy between microbial ecology and molecular biology? – the techniques of molecular biology revolutionized the study of microbial ecology, and vice versa No one can accurately predict which curiosity-based basic research projects (i.e., Thomas Brock's interest in the microbial 6 life in hot springs) will lead to "something important" 6 3 Thomas Brock: – isolated the thermophilic bacterium Thermus aquaticus, source of heat stable DNA polymerase (Taq) used later in polymerase chain reaction (PCR), from a Yellowstone hot spring – later became famous for his textbook – the isolation of the Thermus aquaticus by a microbial ecologist (Brock) led to the development of PCR, one of the most important developments in molecular biology – PCR and related techniques have, in turn, revolutionized the field of microbial ecology 7 7 Thomas Brock 8 8 4 Lower Geyser Basin, Yellowstone, original source of Thermus aquaticus 9 9 Thermus aquaticus 10 10 5 Octopus Spring, Yellowstone (OS-K group) 11 11 PCR movie 12 6 Kary Mullis: – developed the polymerase chain reaction (PCR) using Taq – won the 1993 Nobel Prize for this discovery 13 The PCR Song movie 14 7 Günter Wächtershäuser: – his development of the iron-sulfur world theory and concept of surface metabolism (i.e., on iron pyrite, etc) has been important in the quest to deduce the evolution of the first cells from biochemical reactions on the ancient earth 15 15 Carl Woese: – developed a phylogenetic classification system using the sequence of the 16S (18S in eucaryotes) rRNA genes which are highly conserved and found in ALL cells 16 16 8 Craig Venter (and others): – founded TIGR, ran the Human Genome Project – sequenced multiple Sargasso Sea genomes simultaneously – 1,800 new species, 1.2 million new genes, etc (2004) – currently: J. Craig Venter Institute 17 17 Review of Woese 1990 Three main "Domains" of life: – Archaea, Bacteria, and Eucarya – based upon analysis and comparison of molecular sequences, specifically those of a ribosomal RNA – 16S rRNA is the piece of RNA found in the smaller of the two ribosomal subunits (thus: small subunit rRNA) – found in all organisms – the three "Domains" are more divergent than "Kingdoms" 18 18 9 MICR 331 Microbial Ecology Driscoll Lecture 7 1 1 Simplified historical background: – Traditional: Animal/Plant dichotomy, the ‘aboriginal’ view – Haeckel (1866) ® Three Kingdoms – Animalia, Plantae, Protista – Protista includes a group called Moneres – Moneres (lacking nucleus) were the root of the tree of life – Copeland (1938) ® Four Kingdoms – Animalia, Plantae, Protista, Monera – Whittaker (1959) ® Five Kingdoms – Animalia, Plantae, Protista, Monera, Fungi – parallel development of procaryotic/eucaryotic proposals for the division of life forms made the overall picture confusing – Procaryote theory wasn’t proposed until 1962, thus was not 2 even a basis for Whittaker’s model 2 1 Ernst Haeckel 3 3 Let’s focus on the Monera: – concept originated by Haeckel in the mid-19th century for microbes that were not protists (not eucaryotic microbes) – more recent definition: single-celled procaryotic organisms – generally referred to as ‘bacteria’ – the concept of what a bacterium (or a procaryote) is, proved impossible to define in a taxonomically-sound fashion – in 1977 Woese proposed that there were two types of bacteria: – Eubacteria and Archaebacteria (a new group he proposed) – Classical microbial taxonomy (as available through most of the 20th century) was based upon cytology, morphology and physiology, which are not optimal criteria on their own, but were the best available at the time. This changed in 1977 when Woese based phylogenetic analysis on ribosomal RNA. 4 4 2 Monera and the Five Kingdom system: – why were bacteria (and archaea) placed together in the Monera? – recall: single-celled procaryotic organisms – NB it is also difficult to strictly define ‘single-celled’ – thus, these microbes were placed together in the Monera mostly because they were observed NOT to be eucaryotic – procaryotic was a term used to describe cells that were not eucaryotic, i.e., that were lacking a ‘true nucleus’ – Monera were defined by negative characteristics back to 1866 before being proposed as a Kingdom in 1938: it’s an illogical 19th century category of convenience for microbial misfits – in taxonomy it is not valid to form a group based upon the shared lack of a characteristic 5 5 6 Procaryote theory: The procaryote theory was proposed in 1962 to define bacteria and model the hypothesized evolution of eucaryotes from procaryotes. It was widely accepted before the techniques to test its hypotheses even existed. It fit with the preconceived notions of many. This was well before Archaea were revealed to be a fundamentally-distinct group. The theory was disproven: it should have been entirely abandoned when the hypotheses were found to not be supported by experimental evidence, mostly molecular phylogenetic findings. There is no evidence that eucaryotes evolved from procaryotes. ‘Procaryote’ cannot be defined or used in any valid taxonomic sense. It was a reasonable hypothesis in 1962 and worth putting to the test, but it turned out to be wrong. ‘Procaryote’ was not intended to be used as a phenotypic description; it was an evolutionary hypothesis, thus it is incorrect to now shift its use from evolutionary to phenotypic, because the term is loaded with false evolutionary inference. It persists because it was used for so long before it could be disproven, but now it must be discarded. 6 – See: Pace NR. 2009. Problems with “Procaryote”. J Bacteriol 191:2008-2010. 3 Why couldn’t the Monera be called the Procaryote Kingdom? – Wouldn’t the proposal of a Procaryotic-Eucaryotic two-kingdom dichotomy have solved the taxonomic problems? – No, because the Eucaryotes were divided into several kingdoms – Making a Eucaryote Kingdom would diminish the animal and plant (etc) taxa to sub-kingdom levels – It was also believed (without evidence) that eucaryotes evolved from procaryotes, so putting them at the same taxonomic level was not accepted – This cartoon is fictional! – It’s Haeckel’s 1866 tree! 7 7 Why couldn’t the Five Kingdom system and the ProcaryoticEucaryotic two-kingdom dichotomy co-exist? – There cannot be both five kingdoms AND two kingdoms simultaneously, as the highest divisions of life forms – they are mutually exclusive, not complementary, concepts – A resolution for these two hypothetical arrangements could not be found, and it is obvious why now, because both were proven to be incorrect – neither are supported by the results of analysis of molecular sequences (i.e., 16S rRNA) – both are artificial taxonomies – The Three Domain organisation, as proposed by Woese, has the most relevance today, and the method behind it has very strong and general acceptance among biologists as the most useful universal phylogenetic tool to generate a natural taxonomy. It is an objective and natural taxonomic system, and it eliminates 8 polyphyletic taxa. 8 4 Discovery that stimulated the inevitable change: the Archaea – the finding that the Archaea are phylogenetically distinct from Bacteria proved the Monera to be an artificial taxon and threw everything else into question – also provided tools to discover natural taxonomies – neither the Five Kingdom system nor the Procaryotic-Eucaryotic dichotomy could account for the archaebacteria (Archaea) – interest in the ‘Archaebacteria’, and how they might be related to other organisms, resulted in the need for, and then the actual development of, the proposal of the Three Domains “Wolfe, these things aren’t even bacteria” 9 Carl Woese in 1976, on the rRNA signatures of methanogens (Archaebacteria) 9 No unifying definition of ‘procaryote’: – without a unifying definition, a term such as ‘procaryotic’ cannot be used in taxonomy to group organisms, define them as related – simply stating that procaryotes are not eucaryotes does not unify the procaryotic organisms – eucaryotes possess a membrane-bound nucleus – this is a unifying definition – procaryotes do not possess a membrane-bound nucleus (?) – this is not a unifying definition! – is it even true? – only taxonomically-relevant common characteristics can be used to propose organisms are related – none exist for all ‘procaryotes’ – Norman Pace urges that ‘procaryotic’ only be used in a historical context: it is loaded with so much ‘taxonomic baggage’, even its use as an adjective is too confusing 10 10 5 Some key characteristics of the Three Domains Archaea Bacteria Eucarya Membrane-bound organelles + + + Nuclear membrane -? - + Peptidoglycan cell wall - + - branched chain straight chain straight chain Initiator tRNA a.a. methionine formylmethionine methionine Chloramphenicol resistant sensitive resistant Membrane fatty acids Diptheria toxin sensitive resistant sensitive 16S rRNA dissimilar dissimilar dissimilar Some Archaea seem to have organelles and may have structures similar to a nucleus, Ignicoccus hospitalis has an inner membrane compartment within which 11 it’s DNA is found. Asgard archaea may also have such organelles. 11 12 12 6 From Greening and Lithgow 2020 13 13 14 14 7 From Heimerl et al 2017 15 15 One circular chromosome in ‘procaryotes’ versus multiple linear chromosomes in eucarya? Eucaryotic organisms with single chromosome per haploid set: – nematode Parascaris equorum univalens – ant Mirmecia pilosula Eucaryotic organisms with circular chromosomes: – none known, all have linear chromosomes – organelle genomes (chloroplast, mitochondria) are circular Bacteria with linear chromosomes – Streptomyces coelicolor, S. avermitilis, Rhodococcus sp. strain RHA1, Agrobacterium tumefaciens – Borrelia burdorferi, B. garinii, etc 16 16 8 One circular chromosome in ‘procaryotes’ versus multiple linear chromosomes in eucarya? Bacteria with more than one chromosome – Vibrio species including V. cholerae – Brucella species – Agrobacterium tumefaciens has four replicons: two chromosomes, one circular and one linear, plus two plasmids Archaea with more than one chromosome – Haloarcula marismortui, with two, is the only archaea currently known to have more than one chromosome – But we know comparatively little about Archaea as so many have never been isolated in culture to allow in-depth analysis 17 17 One circular chromosome in ‘procaryotes’ versus multiple linear chromosomes in eucarya? References 1. Kuzminov A. 2014. The precarious prokaryotic chromosome. J Bacteriol 196(10) 1793-1806 2. Nester EW. 2015. Agrobacterium: nature’s genetic engineer. Front Plant Sci doi: 10.3389/fpls.2014.00730 3. Galperin M. 2007. Linear chromosomes in bacteria: no straight edge advantage? Env Microbiol 9(6): 1357-1362 18 18 9 Woese's universal phylogenetic tree (Fig. 1): – distances on the tree correspond to molecular genetic relatedness – i.e., groups close together on the tree have very few nucleotide differences (mutations) in their 16S rRNA genes – relatively few mutations occurred during the time since the groups diverged – 16S/18S sequence comparisons reveal three clusters (Domains) – the sequence differences between the Domains indicate they are distinct and diverged billions of years ago, as depicted below 19 19 Mutations: – mutations cause DNA sequence differences between species – mutations happen at a low rate over time – DNA sequence differences can be correlated with evolutionary distances and time 16S rRNA genes: – molecular chronometer, present in all cells – slowly evolving due to highly conserved ribosome function – secondary structure is critical to function (see next slide) – large enough (~1,600 bp) to have sequence diversity – small enough to make DNA sequencing practical at the time 20 20 10 MICR 331 Microbial Ecology Driscoll Lecture 8 1 1 Mutations: – mutations cause DNA sequence differences between species – mutations happen at a low rate over time – DNA sequence differences can be correlated with evolutionary distances and time 16S rRNA genes: – molecular chronometer, present in all cells – slowly evolving due to highly conserved ribosome function – secondary structure is critical to function (see next slide) – large enough (~1,600 bp) to have sequence diversity – small enough to make DNA sequencing practical at the time 2 2 1 16S rRNA structure 3 3 Ribosomal small subunit 3D structure: proteins in blue, 16S rRNA in orange https://en.wikipedia.org/wiki/16S_ribosomal_RNA 4 4 2 Ribosomes: – made up of many proteins plus three rRNA's – function: translation of mRNA ® protein – contain large (LSU) and small (SSU) subunits – LSU contains two rRNAs (5S and 23S rRNA in bacteria) – SSU contains one rRNA referred to as the 16S rRNA in Bacteria and Archaea or the 18S rRNA in Eucarya – 16S rRNA pairs with sequences on mRNA (ribosomal binding site, or Shine-Dalgarno sequence) to align mRNAs in the ribosome, allowing translation of correct reading frame – LUCA would have had ribosomes – present in all 3 Domains 5 5 Central dogma of molecular biology DNA – RNA - protein nascent protein ribosome mRNA transcription gene ‘ X ’ translation DNA 6 6 3 Translation movie 7 Was the last universal common ancestor (LUCA) a progenote? – a thermophilic anaerobe with complex metabolism? – thermophiles in all three Domains indicate that the LCA at the root of the universal phylogenetic tree was likely thermophilic – progenotes are hypothetical organisms in which cellular information would have been encoded on DNA maintained as unlinked genes – genes would not have been linked in chromosomes, as they are in modern organisms, as the Domains that evolved from the LCA have differing genome structures – a genetic rather than genomic system – genomes evolved after the Domain splits – progenotes evolved from the ‘RNA world’? – "the era of nucleic acid life" (Woese) – Current thinking: LUCA was likely genomic, not a progenote 8 https://www.youtube.com/watch?v=TErdE0rxYyE 8 4 Evolution – time scale Invertebrates Unicellular algae Oxygenic cyanobacteria LUCA Aerobic bacteria Phototrophic bacteria Anaerobic cells then anaerobic bacteria, archaea, eucarya Oxic atmosphere Archaea-Eucarya split? 3.0 2.0 Billion years before present Sexual reporduction 4.0 Autotrophy Photosynthesis Chemical evolution 4.5 Diversity of life Hetertrophy Genomic life Formation of earth Anoxic atmosphere 1.0 0 9 LUCA Physiology Weiss MC, et al. The physiology and habitat of the last universal common ancestor. Nat Microbiol. 2016 Jul 25;1(9):16116. 10 10 5 What happened to the progenotes? – the grouping of genes over time led to genomic life – easier to pass on a complete set of genes to next generation – the last common ancestor (LCA) that diverged into the Bacteria and Archaea/Eucarya branches may have been a progenote RNA world ® progenote LCA ® etc? RNA world ® progenote ® genomic LCA ® etc The last common ancestor: simple or complex? https://www.youtube.com/watch?v=TErdE0rxYyE 11 11 Conclusions: – the procaryote concept is not taxonomically-sound and the procaryote -> eucaryote evolution hypothesis was disproven – the old classification systems not valid for higher-level taxa – models must be dropped when disproven – Woese’s proposal did not change the eucaryotic kingdoms, but revised taxonomy so that the Kingdom is not the highest taxonomic level – the Eucarya (esp. eucaryotic microbes) are poorly classified and revolutionary changes in their taxonomy is occurring because they have mostly adopted molecular phylogenetics – New kingdom-level divisions are being described regularly in all three Domains! 12 12 6 Eucaryal taxonomy: – now (mostly) based on molecular phylogenetic analyses using SSU RNA sequences – unfortunately, some influential eucaryal taxonomists do not accept phylogenetic evidence objectively and that taxonomy should reflect evolution “like genealogies” as proposed by Darwin – their bias is to not discard taxa that are considered to be “useful” historical groupings, even if proven to be polyphyletic – there is still a tendency to refuse to discard the concept that eucaryotes evolved from procaryotes, which is not supported by evidence, and some do not accept the three Domain organization, despite the evidence AND the fact that they have validated Woese’s molecular phylogenetic method by using it! – they prefer to retain “procaryote” as a de facto taxon, and have tried to reinvent that disproven concept (not successfully): See Whitman vs Pace in 2009 (below) 13 13 2006 F D Ciccarelli et al. Science 2006;311:1283-1287. In this paper they use terms Archaea and Archaebacteria, Bacteria and Eubacteria? Not sure what they mean… But clearly 3 domains via genome sequence comparisons Global phylogeny of fully sequenced organisms. The phylogenetic tree has its basis in a cleaned and concatenated alignment of 31 universal protein families and covers 191 species whose genomes have been fully sequenced (14). Green section, Archaea; red, Eukaryota; blue, Bacteria. Labels and color shadings indicate various frequently used subdivisions. The branch separating Eukaryota and Archaea from Bacteria in this unrooted tree has been shortened for display purposes. 14 14 7 2009 Whitman, W. B. 2009. The modern concept of the procaryote. J. Bacteriol. 191:20002005 Pace NR. Rebuttal: the modern concept of the procaryote. J Bacteriol. 2009 Apr;191(7):2006-7. How was this tree that suggests a closer relationship between Bacteria and Archaea even constructed? 15 15 2012 Adl SM, et al. The revised classification of eukaryotes. J Eukaryot Microbiol. 2012 Sep;59(5):429-93. 16 What evidence indicates that eucaryotes evolved from ‘procaryotes’? For this to be true, there must also be a taxonomically-sound definition of ‘procaryote’ 16 8 2016 Hug LA, et al. A new view of the tree of life. Nat Microbiol. 2016 Apr 11;1:16048 17 17 2016 Nasir et al. Arguments Reinforcing the Three-Domain View of Diversified Cellular Life. Archaea 2016 Dec 5;2016:1851865. Distance networks do not support ‘Archaeal Ancestor Scenario’ (AAS) i.e., two vs. three Domains of Life. 18 18 9 2016 Spang and Ettema. The tree of life comes of age. Nat Microbiol. 2016 Apr 26;1:16056 19 19 Asgard Archaea (Loki, Thor, Odin, Hemdall) https://twitter.com/fsantoriello/status/1089288835400228864 20 20 10 Phylogenetic terms 21 21 Orthology 22 22 11 MICR 331 Microbial Ecology Driscoll Lecture 9 1 1 Microbial Diversity 2 2 1 Selected microbial diversity references: 1. 2. 3. 4. 5. 6. 7. 8. Biller SJ, Berube PM, Lindell D, Chisholm SW. 2015. Prochlorococcus: the structure and function of collective diversity. Nat Rev Microbiol. Jan;13(1):13-27. Adékambi T, Shinnick TM, Raoult D, Drancourt M. 2008. Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation. Int J Syst Evol Microbiol. 58(Pt 8):1807-14. Amann RI et al. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143-169. Angert ER et al. 1993. The largest bacterium. Nature 362: 239. Bresler V et al. 1998 Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA. J Bacteriol 180: 5601-5611. Cohan FM. 2002. What are bacterial species? Annu Rev Microbiol 56: 457487. Kassen R & PB Rainey. 2004. The ecology and genetics of microbial diversity. Annu Rev Microbiol 58: 207-231. Konstantinidis KT & JM Tiedje. 2005. Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187: 6258-6264 3 3 9. Konstantinidis KT et al. 2006. The bacterial species definition in the genomic era. Phil Trans R Soc B 361: 1929-1940. 10. Schulz HN, Jorgensen BB. Big bacteria. Annu Rev Microbiol. 2001;55:10537. 11. Taylor et al., 1999. Aerotaxis and other energy-sensing behaviour in bacteria. Annu Rev Microbiol 53: 103-128. (discusses chemotaxis, magnetotaxis, etc) 4 4 2 Recall the central dogma of molecular biology: DNA ® RNA ® protein This can be put into terms that apply to microbial ecology: genetics ® physiology ® ecological niche* ® genetics *site of competition and change in conditions natural selection = microbial evolution Thus, the niche is where mutant strains (especially if they are isolated from each other for a long time) evolve to the point at which they can no longer be considered to be the same species, thus increasing 5 microbial diversity 5 Bacterial species: – in 2004, Bergey's manual listed 4504 species (type strains) – to date, more than 7000 species have been cultured, characterized and accepted – but what is a bacterial species? – first, we need to define "species" What is a species? 6 6 3 What is a species? – in animals, plants, etc – more than a million current eucaryal species known – in general, a pair of organisms mate, and – fertile offspring produced – offspring genomes differ from parents (not clones) because of meiosis, crossing over, etc., – a group of organisms able to interbreed and produce fertile offspring are a species 7 7 How does the ‘species concept’ apply to Bacteria and Archaea? 8 8 4 What are bacterial and archaeal ‘species’? – no firm or completely accepted definition – individual cells are clones – not the products of sexual reproduction – bacterial genetic exchange (not really ‘sex’) involves transfer of pieces of DNA via: – transformation (naked DNA) – transfection/transduction (virus) – conjugation (sex pilus) – not limited to members of the same species and not for the direct purpose of producing offspring – not as much is known about archaeal genetics 9 9 Bacterial & Archaeal Species Systematics 1. 2. 3. 4. 5. Classical bacterial taxonomy DNA-DNA hybridization (DDH) 16S rRNA sequence comparisons Polyphasic taxonomy and new species Whole genome sequence comparison 10 10 5 1. Classical bacterial taxonomy: – physiological & cytological tests for classification purposes – determinative bacteriology (old Bergey's Manuals) – species defined by phenotypic clustering – Gram stain, various biochemical tests – pathogen and symbiont host range (pathovar, biovar) – serological (antibody) and phage typing 11 11 2. DNA-DNA hybridization (DDH) – two organisms may to be considered to be possibly of the same species if their genomes have a minimum of 70% homology via DDH – a useful standard to answer some taxonomic questions – however, within some genera, all of the species are very closely related: – i.e., all Brucella species are within 98% DDH – DDH cannot be used as the sole evidence that two organisms belong to the same species 12 12 6 3. 16S rRNA sequence comparisons: – for two bacterial strains to be considered possibly the same species they must have a minimum 98.7% 16S rRNA gene sequence identity – can be used to distinguish between some species, but more useful for higher level (genus, kingdom, domain) comparisons – other (or multiple) gene sequence comparisons may be more useful for distinguishing between species, subspecies – species cannot be named solely on the basis of 16S rRNA gene sequences obtained from an environmental sample – need evidence that the sequence is from a real cell! – could be artificial chimera – usually requires isolation as a pure culture, but there are exceptions as we will see – 16S rRNA sequencing cannot be used as the sole evidence 13 that two organisms belong to the same species 13 4. Polyphasic taxonomy and new species: – combination of molecular (16S rRNA gene sequences, %DDH) and classical determinative taxonomic data used to characterize strains that have been isolated as pure cultures – for two organisms to be considered to be the same species, they must have >98.7% 16S rRNA identity AND >70% DDH – new organisms that cannot be cultured may be characterized via molecular techniques but will not be accepted as named species until they can be cultured or isolated, such as via cell sorting – new species are accepted by the International Union of Microbiological Societies (IUMS) or the International Committee on Systematics of Prokaryotes (ICSP) and reported via the International Journal of Systematic and Evolutionary Microbiology 14 14 7 5. Whole genome sequence comparison: – compare genomes (min 50 to >500 genes) via amino acid identity (AAI), and/or average nucleotide identity (ANI) – There is a general, but not perfect, correlation between 16S and AAI identities – Konstantinidis and Tiedje 2005, Konstantinidis et al 2006. – compared 16S sequences and AAI identities – comparisons of 175 sequenced genomes – 1752 = more than 30,000 pairwise comparisons 15 15 Konstantinidis, K. T. et al. 2005. J. Bacteriol. 187(18):6258-6264 FIG. 3. Relationships between 16S rRNA, AAI, and taxonomic information for the 175 sequenced genomes 16 8 Currently known diversity of Bacteria – the number of phyla is increasing as new discoveries are made – many ‘candidate’ phyla contain no cultured species – number of bacterial phyla is hard to estimate – currently ~41 accepted (cultured) phylogenetic divisions – approximately 70 candidate phyla in the Candidate Phyla Radiation (CPR) – total is more than 100 phyla as evolutionarily-distant from each other as the old Animal and Plant kingdoms are from each other – is the actual total of bacterial phyla in the range of 1000? http://en.wikipedia.org/wiki/Bacterial_phyla http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=2&lvl 17 =1&lin=f&keep=1&srchmode=1&unlock 17 candidate phyla (CPR) 18 18 9 2016 Hug LA, et al. A new view of the tree of life. Nat Microbiol. 2016 Apr 11;1:16048 1990 19 19 20 10 MICR 331 Microbial Ecology Driscoll Lecture 10 1 1 How many species of Bacteria actually exist? – we will look at diversity of Archaea later in the course – most of the >7000 known bacterial species have been identified via characteristics (biochemical and morphological) that require growth as a pure culture – many are medically-important – how many species would we get if we if we try to calculate the total number of species including those that have not yet been cultured? 2 2 1 Example 1: Soil – look at one gram of a typical soil – molecular techniques reveal the number of species to be approximately 13,000 – thus, many more species in one gram of soil than all of the microbiologists who have ever existed have isolated to date 3 3 Example 2: Insects – there are at least 800,000 insect species (maybe millions) – the gut of each insect contains millions of bacteria and archaea – it is estimated that 10% (80,000) of insect species have bacterial symbionts – symbionts are usually host-specific (unique species) – if only one unique species of symbiont for each of these insect species = 80,000 bacterial species – it is not improbable to hypothesize that there could be at least one unique bacterial species per insect species if we consider the whole insect (not just the gut) – possibly 1 million insect-specific bacterial species – not including possible archaeal symbionts 4 4 2 Perlmutter & Bordenstein. 2020. Microorganisms in the reproductive tissues of arthropods. Nature Reviews Microbiology volume 18, pages 97–111. Published: 06 January 2020 5 5 Habitat vs. Culturability – direct microscopic counts can exceed viable counts by several orders of magnitude – the vast majority of bacterial and archaeal cells observed under the microscope cannot be grown in culture – different proportions are culturable depending on the natural environment the microbial cells were taken from – some may be culturable but are in a viable-but-not-culturable (VBNC) state when we try to isolate them – see below table for commonly-studied microbial habitats – what about environments that have never been studied, or only studied very little i.e., the ocean floor, deep in the earth's crust, the Arctic, Antarctica, etc? 6 6 3 Habitat vs. Culturability Habitat Freshwater Culturability (%) 0.25 Mesotrophic lake 0.1 - 1 Unpolluted estuarine waters 0.1 - 3 Activated sludge 1 - 15 Seawater 0.0001 - 0.1 Sediments 0.25 Soil 0.3 7 7 Culturability Why do some species not grow in any given culture? General reasons: For known species (previously cultured): 1. cells may have entered a non-culturable state (VBNC), or 2. cultivation techniques are known but were not used For uncharacterized species: 3. cells may have entered a VBNC state, or 4. cultivation techniques that would have allowed them to grow were not used 5. we are currently not able to culture them (we may or may not ever be able to grow them): currently not culturable 8 8 4 VBNC State – Viable But Not Culturable – controversial (is it a real phenomenon?) – possibly a dormant state (i.e., like trees during winter) Review on bacterial dormancy: Lennon JT, Jones SE. 2011. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev Microbiol. Feb;9(2):119-30 9 9 Best known VBNC examples: – well-known pathogens Salmonella entertidis, Vibrio cholerae – can enter VBNC state when exposed to certain conditions: – salt water (osmotic shock) – fresh water (osmotic shock) – low temperature (cold shock) – difficult/impossible to revive after such shocks – how do we know they are not dead? 10 10 5 VBNC pathogens – not dead, but deadly: – retain ability to infect and cause disease in host even though they fail to grow in culture under standard conditions – possibly difficult for cells to survive the oxidative burst that may occur when placed in nutrient-rich culture media for revival after having been in a dormant state – is revival in host ‘gentler’ on the cells (slower)? – can VBNC cells be revived in i.e., low-strength media? – Take-home message: chlorinate/disinfect drinking water 11 11 Do we know anything about non-culturable species? 12 12 6 Do we know anything about non-culturable species? – Yes, but only of very few, if they can be identified under a microscope on the basis of unique features and lately via techniques such as FISH – a certain amount about their physiology can be hypothesized from appearance and ecological context – it is now possible to isolate cells via micromanipulation (i.e., cell sorting) for molecular characterization – phylogenetic information may allow some things to be guessed regarding physiology, etc, but cells need to be cultured for indepth physiological characterization – it is now possible, in some cases, to obtain genome sequence information from cells without culturing them, even single cells Next, examples of ‘unculturable’ species about which a fair bit is known 13 13 14 14 7 Epulopiscium fishelsoni – – – – – obligate symbiont in gut of tropical surgeon fish visible to the naked eye: up to 80 x 600 µm shaped like a deflating zeppelin cell volume = 3x106 um3 note that E. coli cell is about 1-2 um long – one million E. coli could fit inside one Epulopiscium! – size restricted by diffusion barrier (nutrients, O2)? 15 15 16 16 8 17 17 18 18 9 E. fishelsoni ecology, phylogeny and reproduction: – appears to be a heterotroph living in a very nutrient-rich environment – may help herbivorous fish host digest food via extracellular enzymes (depolymerization) – physiological potential was revealed via genome sequencing (next slide) – phylogenetically, most closely related to Clostridium species – Firmicutes (Gram positive) division – similar/related species found in other related fish as well as herbivorous land animals – circadian viviparous life cycle – some isolates do not divide by binary fission – use a modified form of endospore formation 19 19 Fig. 6. Metabolic potential of Ca. Epulopiscium and related giant bacteria. Ngugi DK, Miyake S, Cahill M, Vinu M, Hackmann TJ, Blom J, Tietbohl MD, Berumen ML, Stingl U. Genomic diversification of giant enteric symbionts reflects host dietary lifestyles. Proc Natl Acad Sci U 20 S A. 2017 Sep 5;114(36):E7592-E7601. 20 10 21 21 22 22 11 E. fishelsoni genome and DNA replication: – there are many questions about how extremely large cells can function, but let’s just address one: – Question: How can a single copy of a genome control such a large volume of cytoplasm spread over great distances? 23 23 Answer: It can’t. Large cells need to make many copies of the genome that can be distributed throughout the cytoplasm: – extreme polyploidy Hutchison E, Yager NA, Taw MN, Taylor M, Arroyo F, Sannino DR, Angert ER. Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid, giant bacterium Epulopiscium sp. type B. Mol Microbiol. 2018 Jan;107(1):68-80. 24 24 12 Additional Epulopiscium references: – Hutchison E, Yager NA, Taw MN, Taylor M, Arroyo F, Sannino DR, Angert ER. Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid, giant bacterium Epulopiscium sp. type B. Mol Microbiol. 2018 Jan;107(1):68-80. – Ngugi DK, Miyake S, Cahill M, Vinu M, Hackmann TJ, Blom J, Tietbohl MD, Berumen ML, Stingl U. Genomic diversification of giant enteric symbionts reflects host dietary lifestyles. Proc Natl Acad Sci U S A. 2017 Sep 5;114(36):E7592-E7601. – Miyake S, Ngugi DK, Stingl U. Phylogenetic Diversity, Distribution, and Cophylogeny of Giant Bacteria (Epulopiscium) with their Surgeonfish Hosts in the Red Sea. Front Microbiol. 2016 Mar 14;7:285. – Miller DA, Suen G, Clements KD, Angert ER. The genomic basis for the evolution of a novel form of cellular reproduction in the bacterium Epulopiscium. BMC Genomics. 2012 Jun 21;13:265. – Angert ER. DNA replication and genomic architecture of very large bacteria. Annu Rev Microbiol. 2012;66:197-212. E. fishelsoni was the largest known bacterial cell prior to the discovery of 25 Thiomargarita namibiensis 25 – 13 MICR 331 Microbial Ecology Driscoll Lecture 11 1 1 Thiomargarita namibiensis 2 2 1 Thiomargarita namibiensis – giant sulfur bacterium ‘sulfur pearl of Namibia’ – habitat = marine sediments in an upwelling zone – nutrients = NO3- from depths of ocean, H2S from sediments – SOB (sulfide-oxidizing bacterium) – largest bacterium by volume (cell volume up to 200x106 um3) – diameter = 100-750 um, average = 0.5 mm – visible to the naked eye – grow in chains, unattached, in a mucoid sheath several cm long – "string of pearls" – cells appear white, with a very large central ‘vacuole’ – cytoplasm in a narrow band around vacuole – contains white granules of S0 (hence white appearance) 3 3 - see publications by Heide N. Schulz/Schulz-Vogt - Schulz HN, Jorgensen BB. Big bacteria. Annu Rev Microbiol. 2001;55:105-37. 4 4 2 5 Figure 1. Thiomargarita namibiensis. (A) The white arrow points to a single cell of Thiomargarita, 0.5 mm wide, which shines white because of internal sulfur inclusions. Above there is an empty part of the sheath, where the two neighboring cells have died. The cell was photographed next to a fruit fly (Drosophila viriles) of 3 mm length to give a sense of its size. (B) A typical chain of Thiomargarita as it appears under light microscopy. (C) At the left end of the chain there are two empty mucus sheaths, while in the middle a Thiomargarita cell is dividing. (D) Confocal laser scanning micrograph showing cytoplasm stained green with fluorescein isothiocyanate and the scattered light of sulfur globules (white). Most of the cells appear hollow because of the large central vacuole. (E) Transmission electron micrograph of the cell wall [enlarged area in (D)] showing the thin layer of cytoplasm (C), the vacuole (V), and the sheath (S) 5 Largest bacterium Thiomargarita namibiensis beside one of the smallest animals, a Trichogammatidae parasitoid wasp (Trichogamma platneri on a cabbage looper egg). Both the large single bacterial cell on the left and the multicellular wasp are about 0.5 mm in size. 6 6 3 Thiomargarita ecophysiology: – inhabits the upper 10 cm in turbulent marine sediments in upwelling zone – low [O2] in sediments – upwelling periodically brings NO3- to seawater above sediments – the upwelling does not cause the turbulence, they are separate features of this environment 7 7 Zehr JP, Kudela RM. 2011. Nitrogen cycle of the open ocean: from genes to ecosystems. Ann Rev Mar Sci. 3:197-225. 8 8 4 Thiomargarita ecophysiology: – sediments are rich in H2S – H2S is a waste product of sulfate-reducing bacteria (SRBs) that consume organic compounds in anaerobic environments via respiration of SO42– H2S and NO3- do not exist in the same place in significant amounts – microbial metabolism of each creates concentration gradients – Thiomargarita oxidizes sulfide (SOB) and reduces nitrate – needs to be able to travel between the H2S and NO3- zones to survive, but they are non-motile! – passive motility = movement between the H2S and NO3- zones mediated by turbulence – See Figure 9 (Schulz 2001 reference) 9 9 Thiomargarita namibiensis up to 800 mM NO3cytoplasm = thin band 0.5 mm S0 granules - reflect light (white) - also, granules of polyP, glycogen - SOBs turbulent............. upwelling NO3- muck H2S sulfate-reducing bacteria (SRBs) 10 10 5 Fig. 3. T. namibiensis. (A) A single cell of T. namibiensis with many smaller inclusions apart from the large sulfur globules. Inset: higher magnification image of the inclusions. (B) A single cell of T. namibiensis with few smaller inclusions. Inset: higher magnification image of the inclusions. (C) Small inclusions stained dark red for polyphosphate with toluidine blue. Many unstained inclusions can be seen. (D) Small inclusions stained with iodine, showing a dark brown color typical for glycogen. 11 Schulz 2001 Fig 9 Physiology of nitrate-storing sulfur bacteria. (A) Thioploca filaments use their vertical sheaths to commute between sediment surface where they take up nitrate and several cm depths where they reduce H2S and store it as sulfur. (B) Beggiatoa filaments follow the oxygen-sulfide interface (dashed line) up into the overlying water. During times of anoxia, they may survive by using internally stored nitrate as electron acceptor. (C) Thiomargarita can only take up nitrate if the loose sediment gets resuspended. During these times they can easily get into contact with oxygenated water, which they tolerate. When the sediment settles down again, sulfide concentrations become very high. Thiomargarita bridge these periods by surviving on internally stored nitrate. 12 6 Thiomargarita energy generation: – oxidation of H2S, coupled with: – reduction of NO3- (and possibly O2, SO4-) – terminal electron acceptors (TEAs) – in the upper zone: – cells accumulate up to 0.8 M (800 mM) NO3- in the vacuoles – may also reduce O2 as TEA (aerobic respiration) – may be a facultative anaerobe but prefers a low [O2] – the cells carry their own supply of NO3- to the lower zone – allows the cells to oxidize H2S in the lower zone – large size needed for NO3- storage, because cells non-motile? – strategy only possible because of turbulence? – spend more time in H2S zone than other similar species thus need more NO3- storage capacity (and greater size) 13 13 Thiomargarita namibiensis Nutrient flow sea water Energy generation H2S e - NO3- SO42S0 SO42- H2S SRBs muck S0, N2, N2O NH4+ NO3-, SO42- Energy from this reaction powers pumping of protons out of cell (membrane proton gradient drives ATP synthesis). What is the C source? Can it fix CO2? Are these organisms chemolithoautotrophs? 14 14 7 Thiomargarita namibiensis Nutrient flow sea water Energy generation H2S e - NO3- SO42S0 SO42- H2S SRBs muck S0, N2, N2O NH4+ NO3-, SO42- Energy from this reaction powers pumping of protons out of cell (membrane proton gradient drives ATP synthesis). What is the C source? Can it fix CO2? Are these organisms chemolithoautotrophs? 15 15 Thiomargarita namibiensis Nutrient flow sea water Energy generation H2S e - NO3- SO42S0 SO42- H2S SRBs muck S0, N2, N2O NH4+ NO3-, SO42- Energy from this reaction powers pumping of protons out of cell (membrane proton gradient drives ATP synthesis). What is the C source? Can it fix CO2? Are these organisms chemolithoautotrophs? 16 16 8 In the lab: – Thiomargarita can be maintained for a long time in sediment samples – oxidize added H2S – reduce added NO3-, SO42– addition of C sources, such as glucose, acetate did not stimulate growth – C source? – autotroph (chemolithoautotroph)? 0 – S , polyphosphate and glycogen granules – extremely polyploid 17 17 Figure 1 Large bacteria, stained with DAPI, display a similar arrangement of their cellular DNA. (a) Numerous nucleoids found in the peripheral, active cytoplasm of a spherical Thiomargarita namibiensis cell are associated with the plasma membrane. A surface layer focal plane of a small T. namibiensis cell is shown. (b) Half of a large Epulopiscium sp. type B mother cell. This medial section illustrates the peripheral DNA layers of two large internal offspring. Granddaughter cell primordia are seen at the tips of the offspring. At this stage in development, the mother cell DNA has disassembled and is no longer visible. The image of T. namibiensis was kindly provided by Verena Salman. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole. Angert ER. DNA replication and genomic architecture of very large bacteria. Annu Rev Microbiol. 2012;66:197-212. 18 18 9 Thiomargarita metabolic pathways deduced via genomics: – many of the metabolic mysteries were solved via single-cell genomic sequencing (Winkel 2016): – possess genes required for CO2-fixation pathway – thus, they do seem to be chemolithoautotrophic as surmised (C source = CO2) – possess genes needed to utilize O2 and NO3- as terminal electron acceptors for aerobic and anaerobic respiration – possess genes required to utilize H2S and H2 as energy sources (electron donors) 19 19 Winkel M, Salman-Carvalho V, Woyke T, Richter M, Schulz-Vogt HN, Flood BE, Bailey JV, Mußmann M. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions. Front Microbiol. 2016 Jun 21;7:964. 20 20 10 Thiomargarita phylogenetics: – 16S rRNA gene sequence analysis: – within the gamma-Proteobacteria class (i.e., includes E. coli) – Gram negative group – closely related to Thioploca (a motile species) – related to other nitrate-storing sulfur bacteria Beggiatoa – also related to other sulfur/sulfide-oxidizing bacteria (SOBs) such as: – Achromatium oxaliferum, Thiothrix, Thiobacillus – some of which are also very large (next slide) – endosymbionts of the marine animals Riftia pachyptila (worms) and Solemya (bivalves), etc – other Thiomargarita sp. samples have been obtained from the Gulf of Mexico and the Mediterranean (deep sea mud volcano) 21 21 Figure 4 Micrographs of large bacteria. (A) The colorless sulfur bacterium Thiomargarita namibiensis. At the periphery of the cell several sulfur inclusions are visible, whereas the inner part of the cell appears hollow. (B) Scanning electron micrograph of a broken Beggiatoa filament revealing the hollow nature of the cell. (Reproduced from J.M. Larkin and M.C. Henk, 1996). (C) A gut symbiont of surgeonfishes, Epulopiscium fishelsoni, next to a much smaller ciliate. (Reproduced from K.D. Clements and S. Bullivant, 1991). (D) The sulfur bacterium Achromatium oxaliferum containing many large inclusions of calcite. (Reproduced from H.D. Babenzien). (E) Light micrograph of the sulfur bacterium Thioploca araucae, showing three trichomes with sulfur inclusions within a common sheath. (F) Micrograph of a large, marine Oscillatoria (cyanobacterium) isolated from intertidal mats. (Reproduced from C. Castenholz). 22 11 Mediterranean Thiomargarita – Girnth et al 2011 - Summary: A mat-forming population of the giant sulfur bacterium Thiomargarita was discovered at the flank of the mud volcano Amon on the Nile Deep Sea Fan in the Eastern Mediterranean Sea – All cells were of a spherical and vacuolated phenotype and internally stored globules of elemental sulfur. With a diameter of 24–65 µm, Thiomargarita cells from the E. Mediterranean were substantially smaller than cells of previously described populations. – A 16S rRNA gene fragment was amplified and could be assigned to the Thiomargarita-resembling cells by FISH. This sequence is monophyletic with published Thiomargarita sequences, but sequence similarities are only about 94%, indicating a distinct diversification. – In the investigated habitat, highly dynamic conditions favour Thiomargarita species over other sulfur-oxidizing bacteria. – In contrast to T. namibiensis populations, which rely on periodic resuspension from sulfidic sediment into the oxygenated water column, Thiomargarita cells at the Amon mud volcano seem to remain stationary at the sediment surface while environmental conditions change around them due to periodic brine flow. 23 23 Mediterranean Thiomargareta Fig 2 (Girnth) 24 24 12 Mediterranean Thiomargarita – Girnth Figure 2. Morphology of Thiomargarita cells from the Amon mud volcano. – A. Top of a push core from the sulfur band microbial mat presenting sediment-embedded spherical Thiomargarita cells. – B. Phase contrast micrograph of spherical Thiomargarita cells exhibiting highly refractive sulfur inclusions. – C. CLSM image of an individual, FITC-stained Thiomargarita cell featuring a large, central vacuole. Sulfur globules are not visible because FITC staining was performed with cells fixed in ethanol, which is known to dissolve sulfur. – FITC = fluorescein isothiocyanate: fluorescent stain that binds to proteins 25 25 Mediterranean Thiomargarita – Girnth Figure 5. Proposed ecological niche for the Thiomargarita population of the Amon sulfur band in comparison with Thiomargarita namibiensis from Namibian shelf sediments. – At the Amon mud volcano, a short period of exposure to sulfidic brine (A) contrasts a prolonged phase in which cells are in contact with oxygenated bottom water (B). – T. namibiensis cells are buried in sulfidic shelf sediment most of the time (C) and only briefly contact oxygenated bottom water during externally triggered resuspension events (D). – Panel C and D are adapted from Schulz (2006). – This likely explains why T. namibiensis are much larger: they spend more time in the H2S zone of their habitat and need greater NO3- storage capacity 26 26 13 Mediterranean Thiomargareta Fig 5 (Girnth) 27 27 Additional Thiomargarita references: – Winkel M, Salman-Carvalho V, Woyke T, Richter M, Schulz-Vogt HN, Flood BE, Bailey JV, Mußmann M. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions. Front Microbiol. 2016 Jun 21;7:964. – Jones DS, Flood BE, Bailey JV. Metatranscriptomic analysis of diminutive Thiomargarita-like bacteria ("Candidatus Thiopilula" spp.) from abyssal cold seeps of the Barbados Accretionary Prism. Appl Environ Microbiol. 2015 May 1;81(9):3142-56. – Girnth AC, et al. A novel, mat-forming Thiomargarita population associated with a sulfidic fluid flow from a deep-sea mud volcano. Environ Microbiol. 2011 Feb;13(2):495-505. – Grünke S, Felden J, Lichtschlag A, Girnth AC, De Beer D, Wenzhöfer F, Boetius A. Niche differentiation among mat-forming, sulfide-oxidizing bacteria at cold seeps of the Nile Deep Sea Fan (Eastern Mediterranean Sea). Geobiology. 2011 Jul;9(4):330-48. – Kalanetra KM, Joye SB, Sunseri NR, Nelson DC. 2005. Novel vacuolate sulfur bacteria from the Gulf of Mexico reproduce by reductive division in three 28 dimensions. Environ Microbiol. 7(9):1451-60. 28 14 MICR 331 Microbial Ecology Driscoll Lecture 12 1 1 Other Large Nitrate-Storing Sulfur Bacteria (SOBs) Thioploca Beggiatoa Thiomargarita 2 2 1 Other Large Nitrate-Storing Sulfur Bacteria (SOBs) Beggiatoa – Beggiatoa sp. also have a similar H2S-oxidizing (SOB) lifestyle – inhabit the oxygen-sulfide interface (gradient organisms) – follow the O2-H2S interface (dashed line) up into the overlying water and are long, large cells – prefer O2 as terminal e- acceptor, but also store NO3- in large vacuoles for survival under anoxic conditions – heterotrophs – close relatives of Thioploca (next topic) 3 3 Beggiatoa 4 4 2 Beggiatoa 5 5 – – – – – Other Large Nitrate-Storing Sulfur Bacteria Thioploca Thioploca inhabits a niche similar to that of Thiomargarita, has a similar metabolic strategy, but is motile and smaller takes up and stores NO3- in large vacuole while in upper zone generates energy via oxidize of S0 to SO42- in the upper zone and H2S to S0/ SO42- in the lower zone terminal e- acceptor in both zones, NO3- is reduced to NH4+ C source: CO2 or acetate (facultative chemolithoautotrophs) 6 6 3 7 Thioploca sheaths with trichome bundles or individual filaments 7 Thioploca core sample: – tube core (8 cm diameter) collected from a Thioploca bacterial mat in the Peru-Chile oxygen minimum zone – the approximately 1 cm thick mat consists of many individual filaments of giant bacteria. – each filament extends into the sediment and the water; sources of sulfide and nitrate, respectively. – the mat covers nearly 130,000 square km of seafloor – equivalent to the land area of Greece – Census of Marine Life Reveals Abundant Diversity of Microbes. Microbe magazine, January 2011. Image courtesy of Lisa Levin, INSPIRE: Chile Margin 2010, NOAA-OER http://www.microbemagazine.org/index.php/01-2011-current-topics/3025-censusof-marine-life-reveals-abundant-diversity-of-microbes 8 8 4 Thioploca core sample – NB large enough to be visible 9 Thiomargarita vs Thioploca – Thioploca and Thiomargarita are organisms whose energy source and electron acceptor are in spatially-separated zones – Thioploca – can actively move between the H2S and NO3- zones because they are motile – needs to be motile because habitat is not turbulent – is very large, but cells cannot be too large AND motile – Thiomargarita – can move between the H2S and NO3- zones, but only passively via turbulence because they are non-motile – larger size allows it to store more of terminal electron acceptor (nitrate) but precludes motility 10 10 5 Sulfur and Nitrogen Cycles: – we will cover all parts as we go through the course – review at the end – look at the Atlas & Bartha textbook for details of the reactions – Thiomargarita etc were interesting in that the demonstrate microbial roles in each of these cycles: – dissimilatory nitrate reduction – leads to denitrification or nitrite ammonification – H2S oxidation (anaerobic) using NO3- as e- acceptor – H2S oxidation (aerobic) using O2 as e- acceptor – etc? – the sulfide oxidizing bacteria (SOBs) we discussed depend on H2S production (via dissimilatory sulfate reduction) by SRBs (sulfate reducing bacteria) – SRBs, in turn, depend upon SO42- from oxic seawater – these are only the first of many examples! 11 11 Nitrogen cycle Aerobic N2 N2-fixation Nitrification (ammonium oxidation) NH4+ Denitrification Ammonium assimilation n tio ica nif mo Am N2O Anaerobic ammonium oxidation (anammox) Denitrification R-NH2 Nitrite ammonification NO Denitrification Anaerobic NO2- Nitrification (nitrite oxidation) Nitrate assimilation NO2- NO3Denitrification (dissimilatory nitrate reduction) 12 12 6 Sulfur cycle Aerobic H2S Sulfide oxidation (aerobic ) S0 Desulfuration R-SH Sulfur respiration (anaerobic) Chemotrophic & phototrophic sulfide oxidation (anaerobic) S0 Dissimilatory sulfate reduction (anaerobic respiration) Chemotrophic & phototrophic sulfur oxidation (anaerobic) Sulfur oxidation (aerobic) Sulfate assimilation SO42- Anaerobic 13 13 Do we know anything about non-culturable species? – Extreme range in bacterial size and shape – The selective value of shape – CPR phyla TM7 and OP11 – Colourless sulfur bacterium Achromatium oxaliferum – Obligate symbionts of protozoa – Magnetotactic bacteria 14 14 7 Extreme Range in Bacterial Size and Shape – the larger bacteria are incredibly big – largest microbial cell: Thiomargarita namibiensis – the smaller microbes are incredibly small – smallest free-living bacterial cell: Pelagibacter ubique – proportional size difference between the smallest and largest (blue whale) organisms is comparable to a human being relative to the planet Earth – other ultra-small bacteria and archaea are symbionts or obligate parasites/pathogens (reduced genome sizes also) or have only been identified to uncultured phyla (i.e., CPR) – ultra-small bacteria and archaea are found everywhere, including medical implants Luef B, et al. Diverse uncultivated ultra-small bacterial cells in groundwater. Nat Commun. 2015 Feb 27;6:6372. doi: 10.1038/ncomms7372. 15 15 Extreme Range in Bacterial Size and Shape – Pelagibacter ubique – smallest by volume, in terms of known (cultured) species – member of SAR11 clade (first found in Sargasso Sea) – now known as Pelagibacterales (Alphaproteobacteria) – contain proteorhodopsin (light-driven proton pump) – up to 25% of picoplankton (ubiquitous) – Pelagibacter biomass greater than fish biomass in oceans http://schaechter.asmblog.org/schaechter/2015/ 02/the-most-abundant-small-thingsconsidered.html 16 16 8 Fig 1 from: Kevin D. Young. The Selective Value of Bacterial Shape. Microbiol. Mol. Biol. Rev. 2006 70: 660-703 17 17 The Selective Value of Bacterial Shape Young, K.D. 2006. Microbiol. Mol. Biol. Rev. 70: 660-703 Legend for FIG. 1. Variety of prokaryotic shapes. This collage of different cells, unless otherwise stated, is constructed from descriptions and illustrations given by Starr et al. (313) or by Zinder and Dworkin (380). The cells are drawn to scale. Those in the dashed black circle are drawn relative to the 5-µm line. These same cells are included in smaller form in the dashed blue circle to compare their sizes to those of larger bacteria, which are drawn relative to the 10-µm line. (A) Stella strain IFAM1312 (380); (B) Microcyclus (a genus since renamed Ancylobacter) flavus (367); (C) Bifidobacterium bifidum; (D) Clostridium cocleatum; (E) Aquaspirillum autotrophicum; (F) Pyroditium abyssi (380); (G) Escherichia coli (red arrow); (H) Bifidobacterium sp.; (I) transverse section of ratoon stunt-associated bacterium; (J) Planctomyces sp. (133); (K) Nocardia opaca; (L) Chain of ratoon stunt-associated bacteria; (M) Caulobacter sp. (380); (N) Spirochaeta halophila; (O) Prosthecobacter fusiformis; (P) Methanogenium cariaci (NB this is an archaea not a bacteria); (Q) Arthrobacter globiformis growth cycle; (R) gram-negative Alphaproteobacteria from marine sponges (240); (S) Ancalomicrobium sp. (380); (T) Nevskia ramosa (133); (U) Rhodomicrobium vanniellii; (V) Streptomyces sp.; (W) Caryophanon latum; (X) Calothrix sp. The yellow-lined background orb represents a slice of the giant bacterium Thiomargarita namibiensis (290), which is represented to 18 scale with the other organisms. 18 9 MICR 331 Microbial Ecology Driscoll Lecture 13 1 1 Diversity of uncultivated bacteria: TM7 Candidate phylum renamed Saccharibacteria – First proposed as a new phylum in 1998, TM7 16S rRNA sequences were first found in a peat bog but then were found globally – The TM7 phylum appeared to be numerous and diverse – No one was able to isolate TM7 cells for a decade – individual cells isolated from human mouth in 2007 – first cultures were isolated in 2014 (no longer uncultivated!) – TM7 shown to be only the fourth known Gram-positive group via microscopy of sludge prior to success in cultivating these organisms https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&i 2 d=95818&lvl=1&lin=f&keep=1&srchmode=1&unlock 2 1 TM7 3 3 Diversity of uncultivated bacteria: OP11 Candidate phylum renamed ‘Microgenomates’ – – – – First proposed as a new, very diverse phylum, in 1998 Currently multiple (13) phyla in Microgenomates supergroup Original OP11 sequences from Yellowstone’s Obsidian Pool OP11 16S rRNA sequences have subsequently been detected in many places globally i.e., groundwater, human mouth – Partial genome sequence obtained from a VERY TINYsingle cell (ZG1) processed via microfluidic techniques (2011) – Candidatus Microgenomatus auricola SCGC AAA011-E14 – Complete genome sequence reported in 2020 – No cultures have ever been isolated, possibly parasites? https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&i d=1794810&lvl=3&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep 4 =1&srchmode=1&unlock 4 2 ! Phase contrast micrograph 16S rRNA tree * Youssef et al 5 5 Obsidian Pool 6 6 3 Obsidian Pool 7 7 Obsidian Pool 8 8 4 Guerrero Negro, Baja California, Mexico 9 9 Diversity of uncultivated bacteria: TM7 and OP11 References: – He X, McLean JS, Edlund A, Yooseph S, Hall AP, Liu SY, Dorrestein PC, Esquenazi E, Hunter RC, Cheng G, Nelson KE, Lux R, Shi W. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):244-9. – Hugenholtz P, Tyson GW, Webb RI, Wagner AM, Blackall LL. Investigation of candidate division TM7, a recently recognized major lineage of the domain Bacteria with no known pure-culture representatives. Appl Environ Microbiol. 2001 Jan;67(1):411-9. – Luef B, Frischkorn KR, Wrighton KC, Holman HY, Birarda G, Thomas BC, Singh A, Williams KH, Siegerist CE, Tringe SG, Downing KH, Comolli LR, Banfield JF. Diverse uncultivated ultra-small bacterial cells in groundwater. Nat Commun. 2015 Feb 27;6:6372. doi: 10.1038/ncomms7372. Soro V, Dutton LC, Sprague SV, Nobbs AH, Ireland AJ, Sandy JR, Jepson MA, Micaroni M, Splatt PR, Dymock D, Jenkinson HF. Axenic culture of a candidate division TM7 bacterium from the human oral cavity and biofilm interactions with other oral bacteria. Appl Environ Microbiol. 2014 Oct;80(20):6480-9. doi: 10.1128/AEM.01827-14. – – Youssef, N. et al. 2011. Partial Genome Assembly for a Candidate Division OP11 Single Cell from an Anoxic Spring (Zodletone Spring, Oklahoma). Appl Environ Microbiol 10 77(21): 7804–7814. 10 5 Colourless sulfur bacterium Achromatium oxaliferum – – – – – – – diameter up to 100 um (very large cells) oxidizes sulfide (i.e., H2S) compounds, accumulates S0 terminal e- acceptor not understood, perhaps O2 some isolates can fix CO2 distinctive feature: massive CaCO3 (calcite) inclusions buffer (vs H2SO4)? ballast? metabolic waste product? perhaps unculturable because of requirement for other species to provide nutrients in precise concentrations, and/or to remove waste products i.e., grow as part of a community 11 11 Figure 4 Micrographs of large bacteria. (A) The colorless sulfur bacterium Thiomargarita namibiensis. At the periphery of the cell several sulfur inclusions are visible, whereas the inner part of the cell appears hollow. (B) Scanning electron micrograph of a broken Beggiatoa filament revealing the hollow nature of the cell. (Reproduced from J.M. Larkin and M.C. Henk, 1996). (C) A gut symbiont of surgeonfishes, Epulopiscium fishelsoni, next to a much smaller ciliate. (Reproduced from K.D. Clements and S. Bullivant, 1991). (D) The sulfur bacterium Achromatium oxaliferum containing many large inclusions of calcite. (Reproduced from H.D. Babenzien). (E) Light micrograph of the sulfur bacterium Thioploca araucae, showing three trichomes with sulfur inclusions within a common sheath. (F) Micrograph of a large, marine Oscillatoria (cyanobacterium) isolated from intertidal mats. (Reproduced from C. Castenholz). 12 6 Obligate symbionts of protozoa – some fluoresce blue – indicates presence of co-enzyme F420 – only methanogens (Archaea) have co-enzyme F420 13 13 14 14 7 Magnetotactic Bacteria 15 15 Magnetotactic bacteria – most have never been cultured but they can be identified by their magnetotactic behavior and possession of magnetosomes – described as having fastidious nutritional requirements – some species appear to function as multicellular groups – found within diverse phyla: – Nitrospira, Proteobacteria (alpha & delta) – magnetosomes contain magnetite (Fe3O4) or other magnetic materials – can be identified in environmental samples as they contain magnetosomes (chain of magnetic particles) – great diversity observed in appearance of magnetosomes from various species/isolates 16 16 8 Magnetospirillum 17 17 18 18 9 Fig 2 19 19 Multicellular magnetotactic bacteria: – Wenter et al. 2009 – Fig. 2. Ultrastructural analysis of the MMP by SEM at 30 keV. – A. Filamentous surface structures observed in the secondary electron image. – B. The back-scattered electron image reveals the arrangement of single cells within an individual MMP. The rows of magnetosomes can be distinguished based on their high yield of back-scattered electrons which results in a bright signal. – C. Magnetosomes are typically arranged in two or three parallel chains. – D. Close-up of two magnetosome chains. All magnetosome crystals are bullet-shaped, 91 ± 21 nm long and 40 ± 6 nm wide, and show a similar orientation. 20 20 10 Magnetotactic bacteria – Natural history: observations of behaviour – N-hemisphere isolates tend to swim N – S-hemisphere tend to swim S – Q: Why would they tend to display such behaviour? N S 21 21 A: They are not swimming to the actual poles but downward Q: OK, but why? N S 22 22 11 Magnetotaxis is actually MAGNETOAEROTAXIS: – combined magnetotaxis and aerotaxis – to maintain position within a zone of optimal [O2] – initially, only their magnetotactic tendencies were noted because the observations were made under relatively high [O2], not knowing they prefer low [O2] – magnetoaerotactic bacteria sense O2 and sulfide gradients (redox) in addition to magnetic fields – it appears that movement along (parallel to) geomagnetic fields allows them to swim towards the zone with optimal [O2] – microaerobic or anaerobic, depending on the species – tend to swim away from zones where conditions are too oxidizing ([O2] too high) or too reducing (i.e., [S2-] too high) – cells tend to accumulate at the oxic/anoxic transition zone – Thus, they use magnetic field cues as part of their system that directs them to their preferred environmental [O2] 23 23 Chen et al. 2012 Figure 3 24 24 12 Chen et al. 2012 Figure 3 legend: – Diagram showing how magnetotactic bacteria use magnetotaxis to swim to the OATZ in the Northern versus the Southern Hemisphere on Earth – The OATZ is the part of the water column or sediment where oxygen concentrations are preferred by these bacteria. – Dotted black lines show the direction that the bacterium swims. – Solid black lines show the alignment of the magnetite nanoparticles with the Earths geomagnetic field. – Blue circular arrows shows the rotation of the flagellum (clockwise or counterclockwise), which allows the bacterium to swim backward or forward within a water column. – Oxygen and sulfur concentrations are also provided. – Oxygen (O2) is in greater concentration at the top of the diagram, corresponding to the air-water interface. – While the concentration of sulfur (S0) is greater at the bottom of the diagram, corresponding to some depth within the water column or sediment. – NB it is more accurate to think of sulfide (S2-, reduced sulfur), rather than elemental sulfur (S0), as increasing with depth (BD note) 25 25 Magnetotactic bacteria movies Watch in class Magnetotactic bacteria_cuvette http://www.youtube.com/watch?v=l1zKONR81Kg Magnetotactic bacteria_microscope http://www.youtube.com/watch?v=uCKpbIsP-cs Watch on your own http://www.youtube.com/watch?v=WR7j67TABzE https://www.youtube.com/watch?v=3uUL4ooM6KI Building a pyramid: http://www.youtube.com/watch?v=fCSOdQK5PIY Magnetosome structure: http://www.youtube.com/watch?v=uV13UTMNDYw&list=UUb0yCG600t7ZUKIoau_qRmQ&index=5&feature=plcp 26 26 13 References – Magnetotaxis: – Chen, L., Bazylinski, D. A. & Lower, B. H. (2012) Bacteria That Synthesize Nano-sized Compasses to Navigate Using Earth's Geomagnetic Field. Nature Education Knowledge 3(10):30 (http://www.nature.com/scitable/knowledge/library/bacteria-that-synthesizenano-sized-compasses-to-15669190) – Bazylinski DA & RB Frankel. 2004. Magnetosome formation in prokaryotes. Nature Reviews Microbiology 2: 217-230 – Wenter R, Wanner G, Schüler D, Overmann J. 2009. Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments. Environ Microbiol. 11(6):1493-505 27 27 14 MICR 331 Microbial Ecology Driscoll Lecture 14 1 1 Terminal Electron Acceptors in Energy Generation 2 2 1 Chemiosmotic Theory – Peter Mitchell: Nobel Prize in Chemistry 1978 – chemiosmotic theory, oxidative phosphorylation and photophosphorylation – investigating this concept may help you to remember why living cells need a proton motive force 3 3 Chemiosmotic theory references: – Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. 1966. Biochim Biophys Acta 2011 Dec;1807(12):1507-38. – Rich PR. A perspective on Peter Mitchell and the chemiosmotic theory. J Bioenerg Biomembr 2008 Oct;40(5):407-10. 4 4 2 Terminal Electron Acceptors vs Depth – different terminal electron acceptors (TEAs) are available in different niches, generally related to depth, in soil or water – i.e., O2/S2- gradients vs magnetoaerotactic bacteria niches – the reduction (redox) potentials of TEAs decrease with depth, thus energy yields also decrease proportionately – chemotrophs transfer electrons obtained from energy source (organic or inorganic) to the most-oxidizing TEA available – this electron transfer allows them to generate energy via aerobic or anaerobic respiration (see below) – TEA’s availability gradients vary by depth and by distance from the surface of a clump of soil or aquatic floc (see redoxdriven niche partitioning fig by Wright et al. 2012, below) 5 5 Terminal Electron Acceptors vs Depth – The next figure shows how availability of the major nonmetallic terminal electron acceptors differs with depth. In a non-marine environment i.e., soil, the role of sulfate reduction would be minimal, but would be otherwise the same. This is a nice, basic figure: complexities can be built on it, and it can be adapted to different environments and conditions. This is a very important figure for you to understand as it is the most basic representation, and it involves the major biogeochemical cycles 6 6 3 Major terminal electron acceptors: use versus depth O2 CO2 CO2 N2 chemolithoautotrophy O2 à aerobic respiration à à N2 ga Or NO3- à denitrification à sulfide oxidation à H2O marine sediment depth (cm) methane oxidation 0-0.5 0.5-5 SO4- n ic C SO42- à sulfate reduction à CO2 à methanogenesis à à H2S 5-100 à CH4 100 + Terminal electron acceptor use by depth in a marine sediment. Depths are approximate examples only. In nature many factors would influence this distribution, including season. Note this figure only shows “Organic C” electron donors (as used by organotrophs), but the TEA scheme is also true for lithotrophs. Modified from Fig 11.22 Atlas & Bartha, Microbial Ecology: Fundamentals and Applications, 4th ed. 7 Terminal electron acceptors vs. depth in a water column – The next figure shows electron acceptor usage in a water column, which includes metallic TEAs – the redox potentials of available electron acceptors decreases with depth, thus energy yields also decreases proportionately – the easier a compound is to reduce, the better TEA it will be – better TEAs allow cells to pump out more protons (H+) per electron (e-), because e-s fall farther (re. redox) to them – the better TEAs (like O2) are more oxidizing, thus can accept electrons more readily – TEAs are separated by a gradient of better to worse that results from preferential consumption of best possible first – the redox potentials of Mn3+ (to Mn2+) and Fe3+ (to Fe2+) are between those of NO3- and SO428 8 4 Oxygen reduction Aerobic respiration Nitrate reduction Manganese reduction Iron reduction Anaerobic respiration Sulfate reduction Reductive processes in the environment shown by a representative profile (data tracings) from the Black Sea. Electron acceptor profiles (percent of maximum) are plotted versus water depth in meters. The 100% values for each nutrient are approximately: O2 = 300 uM; NO3 = 10 uM; Mn(II) = 20 uM; Fe(II) = 10 uM; and H2S = 20 uM. The zone of Mn/Fe reduction is located between the upper O2-NO3-reduction zone and the lower SO4-reduction zone. Adapted from: Nealson KH, Saffarini D. Annu Rev Microbiol. 1994;48:311-43. See also Fig 11.21 in Atlas & Bartha. 9 9 Wright et al 2012: Redox-driven niche partitioning 10 10 5 Terminal electron acceptors in a Winogradsky column – the Winogradsky column figure shows the presence of aerobes, microaerophiles, and different kinds of anaerobes at distinct depth intervals that differ with respect to available electron acceptors, etc – some organisms can only utilize one TEA while others can utilize more than one, but usually only TEAs that exist in adjacent zones – also shown is consumption of reduced products of anaerobic respiration as electron donors in zones above the zones in which they were produced 11 11 12 6 Major terminal electron acceptors: use versus depth O2 CO2 CO2 N2 chemolithoautotrophy O2 à aerobic respiration à sulfide oxidation à H2O à N2 ga Or NO3- à denitrification à methane oxidation marine sediment depth (cm) 0-0.5 0.5-5 SO4- n ic C SO42- à sulfate reduction à à H2S CO2 à methanogenesis à 5-100 à CH4 100 + Terminal electron acceptor use by depth in a marine sediment. Depths are approximate examples only. In nature many factors would influence this distribution, including season. Note this figure only shows “Organic C” electron donors (as used by organotrophs), but the TEA scheme is also true for lithotrophs. Modified from Fig 11.22 Atlas & Bartha, Microbial Ecology: Fundamentals and Applications, 4th ed. 13 Aerobic respiration – chemotrophic metabolism using O2 as terminal electron acceptor – in-class overview of electron transport chain (ETC) and oxidative phosphorylation (OxPhos) – review basics on your own – NAD+ drives oxidation of electron donor – reducing power (NADH, NADPH, FADH) generated by metabolism (oxidation of energy source) – transferred to ETC, then to terminal electron acceptor (TEA) – O2 ® H2O (via terminal oxidase) – or electrons used in biosynthetic reactions – ETC flow results in proton (H+)/pH gradient – H+ gradient fuels processes like ATP synthesis (OxPhos) 14